Rotors for immunoassays

ABSTRACT

The present invention provides an analyte detection system for detecting target analytes in a sample. In particular, the invention provides a detection system in a rotor or disc format that utilizes a centrifugal force to move the sample through the detection system. Methods of using the rotor detection system to detect analytes in samples, particularly biological samples, and kits comprising the rotor detection system are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application of InternationalApplication No. PCT/US2011/040878, filed Jun. 17, 2011, which claimspriority to U.S. Provisional Application Ser. No. 61/355,847, filed Jun.17, 2010, both of which are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to an analyte detection system fordetecting a target analyte in a sample, e.g. a biological sample. Inparticular, the present invention provides a detection system in a rotorformat that utilizes a centrifugal force to move the sample through thedetection system.

BACKGROUND OF THE INVENTION

One of the most common types of assays used as a rapid point of caretest to detect a particular analyte in a biological sample is a lateralflow strip-based assay. Such assays typically contain a binding partnerfor the analyte of interest coupled to a detectable label (i.e. labeledconjugates) and a porous membrane on which a capture protein (e.g.antibody or antigen) capable of binding the analyte of interest isimmobilized. Labeled conjugates that are commonly used in these types ofassays are antibodies or antigens coupled to gold nanoparticles orcolored latex particles. An analyte present in the sample will bind tothe labeled conjugate to form a complex. The complex continues tomigrate through the porous membrane to the region where the captureprotein is immobilized at which point the complex of analyte and labeledconjugate will bind to the capture protein. The presence of the analyteis then determined by detecting the labeled conjugate in the captureregion of the porous membrane (e.g. by a color change of the captureline).

Although lateral flow strip-based assays have proven useful for rapiddetection of some analytes in the clinical setting, such assays sufferfrom several disadvantages. For example, lateral flow strip-based assaysrequire a series of overlapping porous materials to achieve efficientsample flow through the device. Construction of such devices can becumbersome and somewhat costly depending on the porous materials thatare employed. Also, lateral-strip based assays are inherently limited bytheir sensitivity due to the occurrence of a single binding event andare often limited to a qualitative analysis. In addition, detection ofmultiple analytes in a single sample simultaneously is often difficultto achieve with conventional lateral flow strip-based assays.

Thus, there is a need in the art to develop novel devices and methodsfor the detection of multiple analytes in a sample, particularly abiological sample, that can provide quantitative results as well asqualitative results.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that acentrifugal force can be used to direct fluid sample through radial flowpaths such that the fluid sample contacts reagents and immobilizedbinding partners positioned within the flow paths. A detection deviceemploying radial flow paths obviates the need to use the overlappingporous surfaces of conventional lateral flow devices to achieve propersample flow through the device. In addition, such devices can containmultiple radial flow paths and thus the presence of multiple analytescan be detected simultaneously in a single sample. Accordingly, thepresent invention provides an analyte detection system in a rotor ordisc format that allows for the detection of multiple analytes in abiological sample.

In one embodiment, the detection system comprises a centrifugal force, asample port and a surface, wherein the surface comprises at least onechannel, said channel containing an immobilized capture ligand capableof specifically binding to an analyte in a sample, wherein the sampleport is in fluid communication with said at least one channel, andwherein the centrifugal force is operably connected to the sample portso that when in operation it causes a sample deposited in the sampleport to move through the at least one channel and be in fluid contactwith the capture ligand. In some embodiments, the one or more channelscan be part of a flow path allowing the sample to flow radially outwardwhen a centrifugal force is applied to the system. In other embodiments,the one or more channels can be part of a flow path allowing the sampleto flow along a circular path when a centrifugal force is applied to thesystem. In another embodiment, the surface further contains at least oneabsorbing entity located downstream from the capture ligand.

In some embodiments, the channel further contains a conjugate capable ofbinding to the analyte in the sample to form a complex, wherein thecomplex is captured by the capture ligand. Conjugates present in the oneor more channels of the surface can comprise a binding partnerconjugated to a detectable entity, wherein the binding partner iscapable at specifically binding to a target analyte in a sample. In someembodiments, the binding partner is an antibody or antigen. Thedetectable entity can be a metal particle (e.g. metal nanoparticle ormetal nanoshell), fluorescent molecule, colored latex particle, or anenzyme. In one embodiment, the detectable entity is a gold nanoparticle.

In another embodiment, the channel comprises a first flow path and asecond flow path, wherein said first and second flow paths arepositioned in different planes, and wherein said first and second flowpaths are in fluid communication. The first flow path can comprise animmobilized capture ligand and a conjugate capable of binding to ananalyte in a sample to form a complex that can be captured by thecapture ligand. The second flow path can comprise a substrate region,which comprises a substrate entity capable of interacting with thedetectable entity of the conjugate to produce or amplify a detectablesignal. In one embodiment, the first flow path provides a faster flowthrough than the second flow path when a centrifugal force is applied tothe channel.

In certain embodiments, the surface of the detection system comprises aplurality of channels (e.g., two or more channels), wherein each saidchannel comprises an immobilized capture ligand capable of specificallybinding to an analyte in a sample. In one particular embodiment, eachchannel further comprises a conjugate capable of binding to an analytethe sample to form a complex and wherein the complex is captured by acapture ligand. In some embodiments, each conjugate specifically bindsto a different target analyte in a sample. In one embodiment, each ofthe channels comprises a first and second flow path, wherein said firstand second flow paths are located in different planes and are in fluidcommunication.

In another embodiment, the sample port of the detection system comprisesone or more conjugates capable of binding to an analyte in the sample toform a complex, wherein the complex is captured by a capture ligand. Theconjugates can bind different analytes the sample and may, in someembodiments, contain different detectable entities. In certainembodiments, the surface of the detection system contains a channel,wherein the channel comprises a first capture ligand and a secondcapture ligand, wherein the first capture ligand is located upstreamfrom the second capture ligand. In one embodiment, the first captureligand specifically binds a different analyte than the second captureligand.

In some embodiments, the one or more channels of the detection systemfurther contain a positive or negative control entity. The controlentity can comprise a control binding partner that binds to theconjugate (e.g. binding partner or detectable entity). A detectionsignal from the control entity can indicate proper fluid flow throughthe detection system.

The present invention also provides a kit comprising an analytedetection system of the invention and instructions for using the systemto detect one or more target analytes in a sample. The detection systemsare adapted for use with a centrifugal force and in some embodiments,can be used with conventional centrifuges with appropriate attachments.The kit can further comprise means for collecting samples and buffersfor extracting samples from solid substances.

The present invention includes a method for detecting an analyte in asample. In one embodiment, the method comprises adding the sample to thesample port of an analyte detection system of the invention, applying acentrifugal force to the system, and detecting the binding of theanalyte to the capture ligand. The sample can be a biological sampleisolated from a human or animal subject. In some embodiments, multiple(e.g., two or more) analytes are detected from a single samplesimultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a top view of one embodiment of the analyte detectionsystem of the present invention. A surface (e.g. rotor base or disc)contains a plurality of channels that are in fluid communication with asample port. Each channel contains a conjugate comprised of a bindingpartner conjugated to a detectable entity that can specifically bind toa target analyte present in the sample. At the peripheral edge of eachchannel is a capture ligand capable of binding the analyte-conjugatecomplex. The peripheral edge of each channel can optionally contain acontrol line that indicates sufficient fluid flow through the system.When a centrifugal force is applied to the surface and sample port, afluid sample deposited in the sample port flows radially through thechannels to the periphery of the surface (Hue arrows), and excess fluidis absorbed by absorbent material (absorbing entity) positioneddownstream of each capture ligand. The surface can optionally contain ablood separator material, which allows plasma from a blood sample topass into the system while retaining cellular material in the sampleport.

FIG. 2 illustrates a top view of another embodiment of the analytedetection system of the present invention. A surface (e.g. rotor base ordisc) contains a single channel in fluid communication with a sampleport. The sample port contains multiple conjugates, each of which iscomprised of a binding partner conjugated to a detectable entity. Theconjugates are capable of specifically binding to different targetanalytes present in the sample. The peripheral edge of the surfacecontains multiple capture ligands capable of binding particularanalyte-conjugate complexes. When a centrifugal force is applied to thesurface, a fluid sample deposited in the sample port contacts themultiple conjugates. If the particular analyte to which the conjugatebinds is present in the sample, an analyte-conjugate complex is formedand flows radially through the channel to the periphery of the surface(blue arrows). Capture ligand immobilized on the peripheral edge of thesurface will capture the analyte conjugate complex. Multiple analytes inthe sample may be detected by employing different capture ligands thatspecifically bind to the different analyte-conjugate complexes. Excessfluid is absorbed by absorbent material (absorbing entity) positioned atthe end of the peripheral flow path (e.g., downstream of all captureligands). A control line, which indicates sufficient fluid flow throughthe system, may optionally be positioned at the end of the peripheralflow path upstream of the absorbing entity. Preferably, a waterimpervious material (block) is positioned between the end of theperipheral flow path and the point where the channel delivers fluid tothe peripheral edge of the surface. The surface can optionally contain ablood separator material, which allows plasma from a blood sample topass into the system while retaining cellular material in the sampleport.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery that multipleanalytes in a sample can be detected simultaneously by utilizing acentrifugal force to direct a fluid sample through multiple radial flowpaths. Accordingly, the present invention provides an analyte detectionsystem in a rotor or disc format that can provide qualitative orquantitative detection of a range of analytes in test samples.

In one embodiment, the analyte detection system comprises a centrifugalforce, a sample port, and a surface, wherein the surface comprises atleast one channel, said channel containing an immobilized capture ligandcapable of specifically binding to an analyte in a sample, wherein thesample port is in fluid communication with said at least one channel,and wherein the centrifugal force is operably connected to the sampleport so that when in operation it causes a sample deposited in thesample port to move through the at least one channel and be in fluidcontact with the capture ligand.

The surface is preferably a disc (e.g. circular) or rotor shape andcontains at least one channel. In some embodiments, the surface containsa plurality of channels, for instance, two or more, three or more, fouror more, five or more, six or more, seven or more, eight or more, nineor more, or ten or more channels. The number of channels on the surfacecan be adjusted to accommodate the number of analytes that are to bedetected simultaneously. The total number of channels is limited only bythe area of the surface, which in turn is limited by dimensions ofconventional centrifuges. In certain embodiments, the surface isproportioned to fit within conventional centrifuges.

The surface can be constructed of a wide variety of materials, includinghut not limited to, plastic, acrylic resin, silica plate, metal plate,polycarbonate, polypropylene, ceramic material, or a laminated or coatedmaterial. The surface should be constructed of a material that is ableto withstand a centrifugal force of at least 1,600 g. One or morechannels can be formed in the surface material by creating depressionsusing conventional techniques. Alternatively, the channels can be formedby affixing pre-formed channels (e.g. plastic strips) to the surface.The channels are preferably arranged radially on the surface such thatthe channels provide a flow path from the sample port, which can belocated at the center of the surface, to the peripheral edge of thesurface. For instance, in one embodiment, the one or more channels arepart of one or more flow paths that allow the sample to flow radiallyoutward when a centrifugal force is applied to the sample port andsurface (see, e.g., FIG. 1). In another embodiment, the surfacecomprises a single channel that is part of a flow path allowing thesample to flow along a circular path (e.g. around the circumference ofthe surface) when a centrifugal force is applied to the sample port andsurface (see, e.g., FIG. 2).

The sample port is in fluid communication with one or more channelspresent on the surface. As used herein, “fluid communication” refers tothe ability of a liquid to flow or travel between two materials orsurfaces. Fluid communication can be established between two porousmaterials or between a porous material and anon-porous material. In thelatter situation, the non-porous material can form a channel or conduitby which fluid can flow by capillary action to establish fluidcommunication between the non-porous material and the porous material.In some embodiments, sample deposited in the sample port flows into oneor more channels forming a flow path from the center of the surface tothe periphery or outer edge of the surface.

In certain embodiments, the sample port is positioned at the center ofthe surface. The sample port provides an entry point for liquid sampleto be applied to the detection system. As discussed in more detailbelow, liquid sample applied to the sample port can enter one or moreradial flow paths when a centrifugal force is applied to the sampleport. In some embodiments, the sample port comprises a sample reagentselected from the group consisting of blocking agents, neutralizingagents, buffers, detergents, antimicrobials, and combinations thereof.One or more of the sample reagents may be dried into a pad positioned inthe sample port. The pad can be manufactured from one of severalmaterials, including but not limited to, polyester, polyacrylic, otherpolymeric materials, or glass fiber.

In one embodiment, the sample port comprises a blocking agent. A“blocking agent” is an agent that prevents the non-specific associationof proteins present in the sample with the conjugates, the immobilizedcapture ligands, and/or target analytes. Blocking agents are typicallyproteins themselves and can include, but are not limited to, bovineserum albumin, casein, gelatin, ovalbumin, gamma-globulins, and IgG fromnon-immunized animals. In another embodiment, the sample port comprisesa neutralizing agent. A “neutralizing agent” is an agent that reducesthe chemical reactivity of at least one interfering species. Aninterfering species can be a biological molecule or other compoundpresent in a sample that exhibits anon-specific binding affinity to thedetectable entity in the conjugate, Non-limiting examples ofneutralizing agents include alkylating agents, such as iodoacetamide,iodoacetate, N-ethylmaleirnide, PEG-maleimide, ethlymethanesulfonate,4-vinylpyridine, nitrogen mustards, nitrosourea compounds, dacarbazine,and temozolomide. Neutralizing agents are described in detail in WO2010/006201, filed Jul. 9, 2009, which is herein incorporated byreference in its entirety.

The sample port can comprise other various sample reagents including,but not limited to, buffers for maintaining the pH of the sample,detergents to enhance fluid flow, accelerants for enhancing the rate ofimmunoreactions, and antimicrobials to prevent biological contamination.Non-limiting examples of suitable buffers include iris, Hepes,imidazole, phosphate and other standard buffers typically used inlateral flow assays. Suitable detergents that may be used include, butare not limited to, Tween-20, Triton X-100, saponin, zwittergents basedon sulfobetaines, CHAPS, octyl glucosides, and lauryl sulfates. Suitableaccelerants that may be incorporated into the sample port include, butare not limited to, polyethylene glycols, polyvinyl alcohols, andpolyvinylpyrrolidones. Exemplary antimicrobials that may be incorporatedinto the sample port include sodium azide, thimerosal, Proctins,antibiotics (e.g. Aminoglycosides, Ansaniycins, Carbacephem,Carbapenems, Cephalosporins, Macrolides, Monobactams, Penicillins,Quinolones, Sulfonamides, and Tetracyclines), antivirals (e.g.amantadine, rimantadine, pleconaril, acyclovir, zanamivir, andoseltamivir), antifungals (e.g. Natamycin, Rimocidin, Filipin, Nystatin,Amphotericin B, Candicin. Imidazoles, Triazoles, Allylamines, andEchinocandins), and antiparasitics (e.g. Mebendazole, Pyrantel pamoate,Thiabendazole, Diethycarbazine, Niclosamide, Praziquantel, Rifampin,Amphotericin B, and Melarsoprol), One of ordinary skill in the art canselect other appropriate antimicrobials, buffers, accelerants, anddetergents based on the particular sample type to be screened and theparticular target analytes to be assayed without undue experimentation.

In certain embodiments, the sample port comprises a blood separatormaterial. A blood separator material can be a filter material with poresizes that allow plasma in a whole blood sample to pass through thefilter and enter the detection system while cells and cellular debrisare retained on the filter. For instance, filter materials with a micronrating of 8 μm or lower may be used. Blood separating materials areavailable commercially from Pall (Vivid), MDI (FR1 and FR2) and Whatman(Fusion 5).

In some embodiments, the one or more channels present on the surfacecontain an immobilized capture ligand. The capture ligand is capable ofspecifically binding to a target analyte in a sample. The capture ligandcan be a biological macromolecule, such as an antibody or a regionthereof (e.g., Fv, single chain, CDR, antibody expressed in phagedisplay, etc.), a receptor, a ligand, a polynucleotide, an aptamer, apolypeptide, a polysaccharide, a lipopolysaccharide, a glycopeptide, alipoprotein, or a nucleoprotein. In some embodiments whole cells,bacteria, or viruses can be immobilized to serve as the capture ligands.The capture ligand can be the same type of molecule as the bindingpartner in the conjugate, but preferably interacts with the targetanalyte at a location distinct from that as the binding partner. By wayof example, the binding partner and the capture ligand can both beantibodies that recognize a target analyte, but the epitope to which thebinding partner binds the target analyte is separate from the epitope towhich the capture ligand binds the target analyte.

In one particular embodiment, the capture ligand is immobilized in thechannel at a point downstream of the sample port. As used herein,“downstream” refers to the direction of fluid flow toward the end of thedetection system and away from the site of sample application. In someembodiments, downstream refers to fluid flow radially outward from thecenter of the surface to the peripheral edge of the surface. “Upstream”refers to the direction of fluid flow away from the end of the detectionsystem and toward the site of sample application. In some embodiments,the surface comprises a plurality of channels, said channels eachcomprising an immobilized capture ligand capable of specifically bindingto an analyte in a sample. See, e.g., FIG. 1. Each capture ligand canspecifically bind to a different analyte in the sample such that onedetection system can be used to simultaneously detect multiple analytespresent in a single sample. Thus, each radial flow path formed by eachchannel can contain an immobilized capture ligand capable of bindingspecifically to a particular analyte in a sample.

In other embodiments, different capture ligands can be immobilized atdifferent points on the peripheral edge of the surface. See, FIG. 2. Forinstance, the surface can comprise a single channel that contains afirst capture ligand and a second capture ligand, wherein the firstcapture ligand is located upstream from the second capture ligand. Insome embodiments, the surface can comprise a plurality of captureligands arranged sequentially along the sample flow path. In suchembodiments, the channel can form part of a flow path allowing thesample to flow along a circular path (e.g. around the circumference ofthe surface) when a centrifugal force is applied to the sample port andsurface. In one embodiment, the capture ligands are capable ofspecifically binding different target analytes present in the sample. Byway of example, the first capture ligand is capable of specificallybinding a first target analyte and the second capture ligand is capableof specifically binding a second target analyte. In another embodiment,the different sets of capture ligands are capable of specificallybinding the same target analyte and each set of capture ligands reflectsa particular concentration at analyte. For instance, a first captureligand can be Immobilized at a low concentration, a second captureligand can be immobilized at a medium concentration, and a third captureligand can be immobilized at a high concentration. A sample containing amedium concentration of the target analyte will contact and bind thefirst capture ligand. Having saturated the first capture ligand, excesstarget analyte will flow to and bind the second capture ligand. Sincethe target analyte in the sample will have bound to either the first orsecond capture ligand, no target analyte remains to flow to and bind thethird capture ligand. Thus, the device will give a read out at the firstand second capture ligands, but not the third capture ligand. Theconcentration of the target analyte in the sample can then be calculatedbased on the binding of the analyte at the different sets of captureligands.

The capture ligands can be immobilized directly to the surface materialor channel material. Alternatively, the capture ligands can beimmobilized to a porous material, which in turn is affixed to thesurface or channel material, A “porous” material refers to a materialcontaining a plurality of interstices or pores through which liquideasily flows. The porous material can be made from natural or syntheticsubstances. Suitable porous materials for use in the detection system ofthe present invention include, but are not limited to, nitrocellulosicmaterial, acrylic material, PVDF, polyethylene material (e.g. Porex®),nylon, cellulose acetate, polyester material, PES material, orpolysulfone material. Other appropriate porous materials that can beused in the detection systems of the invention are known to thoseskilled in the art. The capture ligands can be immobilized to variousmaterials (e.g. porous materials) by a variety of procedures. Thecapture ligands can be striped, deposited, or printed on the materialfollowed by drying of the surface to facilitate immobilization.Immobilization of the capture ligands can take place through adsorptionor covalent bonding. Depending on the nature of the material (e.g. typeof material), methods of derivatization to facilitate the formation ofcovalent bonds between the material and the capture ligand can be used.Methods of derivatization can include treating the material with acompound, such as glutaraldehyde or carbodiimide and applying thecapture ligand. Other physical, chemical, or biological methods ofimmobilizing a macromolecule or other substance either directly orindirectly to a material are known in the art and can be used toimmobilize the capture ligand to the surface material, channel material,or porous material of the detection system. In embodiments which utilizeporous materials, the porous material on which the capture ligand isimmobilized may be treated with a blocking agent, sun as bovine serumalbumin or other blocking agent as described herein.

In certain embodiments, the one or more channels present on the surfacefurther comprise a conjugate capable of binding to the analyte in thesample to form a complex, wherein the complex is captured by the captureligand. The conjugate comprises a binding partner conjugated or linkedto a detectable entity. The binding partner can be any entity that iscapable of specifically binding to a target analyte. In someembodiments, the binding partner is a biological macromolecule,including but not limited to an antibody or a region thereof (e.g., Fv,single chain, CDR, antibody expressed in phage display, etc.), areceptor, a ligand, a polynucleotide, an aptamer, a polypeptide, apolysaccharide, a lipopolysaccharide, a glycopeptide, a lipoprotein, ora nucleoprotein. In one embodiment, the binding partner is an antibody.In another embodiment, the binding partner is an antigen.

As used herein, “detectable entity” is an entity that is capable ofproducing a detectable signal under a particular set of conditions. Inone embodiment, the detectable entity is an entity that exhibitswavelength selective absorption in the ultra-violet, visible, or nearinfrared electromagnetic spectrum and scatters incident radiation. Forinstance, the detectable entity can be a metallic nanoparticle ormetallic nanoshell, Various types of metallic nanoparticles that can becoupled to the binding partner include, but are not limited to, goldnanoparticles, silver nanoparticles, copper nanoparticles, platinumnanoparticles, cadmium nanoparticles, composite nanoparticles (e.g.silver and gold or copper and silver), and gold hollow spheres. In someembodiments, the detectable entity is a gold nanoparticle. Additionally,metal nanoshells as described in U.S. Pat. No. 6,699,724, which isherein incorporated by reference in its entirety, can also be used asthe detectable entity. Metal nanoshells are particles comprised of adielectric core and a metallic coating that have a defined core radiusto shell thickness ratio. The core can be comprised of a variety ofmaterials including silicon dioxide, gold sulfide, titanium dioxide, andpolystyrene. Suitable metals for the shell include gold, silver, copper,platinum, palladium, lead, and iron. Gold-coated silica nanoshells orsilica-coated gold shells are preferred in some embodiments.

In some embodiments, the detectable entity is an enzyme. Preferably theenzyme is capable of converting a substrate into a detectable product,e.g., colored, fluorescent, or chemiluminescent product. Non-limitingexamples of enzymes that are suitable for conjugation to the bindingpartner include alkaline phosphatase, horseradish peroxidase,beta-galactosidase, beta-lactamase, galactose oxidase, lactoperoxidase,myeloperoxidase, and amylase. In another embodiment, the detectableentity is a metallic nanoparticle conjugated to an enzyme.

Other molecules, such as fluorescent molecules (e.g. fluorescein,Texas-Red, green fluorescent protein, yellow fluorescent protein, cyanfluorescent protein, Alexa dye molecules, etc.), that are known to thoseskilled in the art can be used as detectable entities in the conjugatesof the invention. In some embodiments, the detectable entity is acolored latex particle as described, for example, in U.S. Pat. No.4,837,168, which is herein incorporated by reference. In one embodiment,the conjugate is an antibody-nanoparticle conjugate. In anotherembodiment, the conjugate is an antibody-colored latex particleconjugate. In another embodiment, the conjugate is anantigen-nanoparticle conjugate. In still another embodiment, theconjugate is an antibody-enzyme conjugate. In yet another embodiment,the conjugate is an antibody-enzyme-nanoparticle conjugate. The enzymein such conjugates can be alkaline phosphatase, horse radish peroxidase,or β-galactosidase.

Methods of conjugating a detectable entity (e.g. metallic nanoparticles,metallic nanoshells, colored latex particles, fluorescent molecules, andenzymes) to a binding partner are well known in the art. One such methodfor coupling a metallic nanoparticle or metallic nanoshell to a bindingpartner is by passive adsorption. This method involves adjusting the pHof the metal colloid solution to a pH at which the protein or otherbinding partner to be labeled has a positive charge, mixing the metalcolloid solution with the binding partner solution, and centrifuging theresultant mixture. The labeled binding partner (e.g. protein) is thenobtained by removing the supernatant and resuspending the precipitate.Other methods of conjugating macromolecules to detectable entities areknown to the skilled artisan, who can select the proper method based onthe type of desired detectable entity to be used and the type ofmacromolecule to be labeled. In some embodiments, the binding partnercan be coupled to the detectable entity indirectly through a largercarrier molecule or protein. Such indirect coupling is particularlyuseful when the binding partner is small, such as a hormone, drug, orother small molecule less than 10 kD. For example, the detectable entity(e.g., gold nanoparticle) coupled to streptavidin can be conjugated tobiotinylated binding partners (e.g., antigens or antibodies).Preferably, the carrier protein is not capable of specific interactionwith the target analyte. In some embodiments, the binding partner iscoupled to the detectable entity to form the conjugate prior todeposition of the conjugate on the surface or channel material.

In certain embodiments, the one or more channels present on the surfacecomprise a first flow path and a second flow path Preferably, the firstand second flow paths are located in different planes and are in fluidcommunication. For instance, the first flow path of the channel can belocated in a top plane, while the second flow path of the channel can belocated in a bottom plane positioned directly underneath the top plane.Thus, fluid flow through such a channel will be split into two flowpaths when a centrifugal force is applied to the system. Devices withsimilar split flow paths are described in PCT/US2010/026948, filed Mar.11, 2010, which is herein incorporated by reference in its entirety. Thedual flow paths allow for easy amplification of the detection signalwithout the need for multiple reagent application steps or washes. Inone embodiment, the first flow path provides a faster flow through thanthe second flow path when a centrifugal force is applied to the one ormore channels. For instance, in some embodiments, the fluid flow throughthe first and second flow paths is controlled by employing differentmaterials (e.g., different porosity membranes, presence of detergents)in the two flow paths or altering the lengths of each of the flow paths.In other embodiments, the fluid flow through the first and second flowpaths is controlled by applying different, sequential centrifugal forcesto the flow paths—i.e. employing a lower speed spin followed by a higherspeed spin to the detection system. In still other embodiments, thefirst and second flow paths may contain different materials such thatfluid flow through the second flow path is slower than fluid flowthrough the first flow path. Combinations of different materials in thetwo flow paths and sequential application of increasing centrifugalforces to the detection system to achieve different flow rates throughthe first and second flow paths are also contemplated.

In one embodiment, the first flow path comprises an immobilized captureligand and a conjugate capable of binding to an analyte in the sample asdescribed herein. In another embodiment, the conjugate is positionedupstream of the capture ligand. In some embodiments, the first flow pathis in fluid communication with the sample port such that liquid sampledeposited in the sample port will flow into the first flow path when acentrifugal force is applied to the system.

In another aspect of the invention, the second How path comprises asubstrate region. The substrate region can contain a pad in which one ormore substrate entities (e.g. enzyme substrates, amplifying reagents,polymers, etc.) are dried. In some embodiments of the invention, theconjugate located in the first flow path comprises a binding partnerconjugated to a detectable entity and the substrate region comprises asubstrate entity capable of interacting with the detectable entity toproduce a detectable signal. By way of illustration, if the conjugatecomprises an enzyme as the detectable entity, the substrate region cancomprise a substrate for that enzyme wherein a colored, fluorescent, orchemiluminescent substance is produced from the substrate after reactionwith the enzyme. The specific substrate will depend upon the type ofenzyme used as the detectable entity and the type of signal desired(e.g. color change or fluorescent signal). Some examples of suitablesubstrates include, but are not limited to,2,2′-Azino-bis-(3-ethylbenziazoline-6-suifortic acid) (ABTS),3-Amino-9-ethylcarbazoie (AEC), 5-bromo-4-chloro-3-indolylphosphate/tetranitroblue tetrazolium chloride (BCIP/NBT),5-bromo-4-chloro-3-indolyl phosphate/tetranitroblue tetrazolium(BCIP/TNBT), Lumiphos®, 3,3′-Diaminobenzidine (DAB),3,3′,5,5′-Tetramethylbenzidine (TMB),5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), phosphastesindoxyls substituted at various positions in combination with a varietyof tetrazolium dyes, naphthol phosphates in combination with Fast dyes,4-CN, cobalt-DAB, and Gold-DAB, Various chromogenic, fluorogenic, andchemiluminescent substrates are commercially available for standardenzymes, such as alkaline phosphatase and horseradish peroxidase. Suchcommercially available enzyme substrates can be used in the detectionsystem of the invention.

In other embodiments of the invention, the conjugate in the first flowpath comprises a binding partner conjugated to a detectable entity andthe substrate region comprises a substrate entity capable of interactingwith the detectable entity to amplify the signal from the detectableentity. For instance, in one embodiment, the detectable entity is a goldnanoparticle and the substrate entity is silver nitrate. In anotherembodiment, the detectable entity is a metallic nanoparticle and thesubstrate entity is 3,3′,5,5′-Tetramethylbenzidine (FMB) or anindigo-containing product. The substrate region may comprise one or moreamplifying reagents that intensify the signal from the detectable entity(e.g. metal nanoparticles or metal nanoshells). Such amplifying reagentsinclude, but are not limited to, silver nitrate, silver acetate, silvercitrate, osmium tetroxide, diaminobenzidine, tetrazolium dyes,peroxidase reaction product of 3,3′,5,5′-Tetramethylbenzidine (17 MB),alkaline phosphatase reaction product of BLIP or any otherindigo-containing product (e.g. 3-IP or any of the other substitutedindoxyl phosphates). In another embodiment, the substrate entityproduces a product that enhances the color of metallic nanoparticles.The product may be formed enzymatically. For instance, in oneembodiment, the detectable entity is a metallic nanoparticle conjugatedto an enzyme and the substrate entity is a substrate of the enzyme. Insome embodiments, the detectable entity is a gold nanoparticle-enzymeconjugate and the substrate entity is 3,3′,5,5′-Tetramethylbenzidine(FMB) or an indigo-containing product (e.g. BCIP or 3-IP).

The substrate region can comprise one or more additional reagents thatact to slow the flow of fluid through the second flow path. In oneembodiment, the substrate region comprises one or more slowly dissolvingpolymers (e.g. dissolution retardants); such as polyvinylpyrrolidone,polyvinyl alcohol, polyethylene glycol, ethylcellulose,hydroxypropylmethylcellulose, Eudragit® and equivalent polymethacrylateproducts, hydroxypropylethylcellulose and hydroxypropylcellulose, orvarious guar gums, to retard the dissolution of dried substrate oramplifying reagent present in the substrate region, thus prolonging thedelivery of the substrate or amplifying reagent to the capture ligandlocated in the first flow path. In some embodiments, higher molecularweight polyvinylpyrrolidones are preferred. In still another embodiment,the substrate region is gelled with calcium-alginate and is fluidizedwith EGTA contained in the sample port.

In certain embodiments, the second flow path is in fluid communicationwith the sample port of the detection system such that liquid sampleflows into both the first and second flow paths when centrifugal forceis applied to the system. In other embodiments, the second flow pathcomprises a sample entry region positioned downstream of the sample portand upstream of the substrate entry region. The sample entry region canbe in fluid communication with the first flow path. In such embodiments,when centrifugal force is applied to the system, fluid sample depositedin the sample port enters the first flow path and subsequently entersthe second flow path through the sample entry region. In someembodiments, the second flow path further comprises a substrate entryregion positioned downstream of the substrate region. The substrateentry region can also be in fluid communication with the first flowpath. In certain embodiments, fluid communication between the substrateentry region and the first flow path is established through asemi-permeable membrane or an air gap that fills upon liquid absorption.Preferably, fluid from the second How path re-enters the first How pathupstream of the capture ligand through the substrate entry region.

Thus, in certain embodiments in which each channel of the detectionsystem of the invention comprises a first and second flow path, acentrifugal force applied to the system will cause liquid sampledeposited in the sample port of the system to flow radially into thefirst and second flow paths of each channel. Fluid and dissolvedreagents, such as enzyme substrates and amplifying reagents, from thesecond flow path will be delivered to the immobilized capture ligandslocated in the first flow path at a later point in time than fluid inthe first flow path. For instance, in one embodiment, when a centrifugalforce is applied to the detection system, liquid sample placed in thesample port will flow radially into the first and second flow paths ofeach channel. Liquid sample entering the first flow path will contactthe conjugate and any target analyte present in the sample will form acomplex with the conjugate. The fluid will continue to flow downstreamthrough the first flow path to the immobilized capture ligand, where thetarget analyte-conjugate complex will bind to the capture ligand. Thefluid sample entering the second flow path will contact and solubilizethe substrate entity (e.g. enzyme substrate or amplifying reagent) inthe substrate region and flow downstream re-entering the first flow paththrough the substrate entry region. The dissolved substrate entity willthen contact the capture ligand and any bound target analyte-conjugatecomplex producing a detectable signal or enhancing a detectable signal.The flow rates through the first and second flow paths of each channelcan be controlled by applying different strength centrifugal forces. Forexample, in one embodiment, a lower strength centrifugal force willcause liquid sample to flow through the first flow path to the captureligand, while a higher strength centrifugal force will result in theflow of fluid through the second flow path, which is delayed due to thepresence of viscous materials or valves that preferentially open athigher speeds.

In one embodiment, the surface comprises a plurality of channels (e.g.two or more), wherein each channel comprises a conjugate capable ofbinding to an analyte in the sample to form a complex and wherein thecomplex is captured by a capture ligand. In certain embodiments, eachconjugate is capable of binding to a different analyte in the samplesuch that multiple analytes can be detected simultaneously. Thedetectable entities in each conjugate can be the same or different. Forinstance, the detectable entities in each conjugate may be a goldnanoparticle. Alternatively, the detectable entity in a first conjugatemay be a gold nanoparticle, while the detectable entity in a secondconjugate may be a silver nanoparticle. In some embodiments, all thedetectable entities in a single detection system are of the same type(e.g. metal nanoparticles/nanoshells, fluorescent molecules, coloredlatex particles, or enzymes).

In another embodiment, the sample port comprises at least one conjugatecapable of binding to an analyte in the sample to form a complex,wherein the complex is captured by a capture ligand downstream of thesample port. In certain embodiments, the sample port comprises two ormore conjugates capable of binding a target analyte. In preferredembodiments, each conjugate is capable of binding a different targetanalyte in the sample. The detectable entities in each of the conjugatescan be the same or different.

Conjugates can be deposited directly on the surface or channel materialor can be deposited on a porous material, which in turn is affixed tothe surface or channel material. Porous materials on which conjugatesare deposited may further comprise one or more excipients to stabilizethe conjugates. Such excipients will depend on the type of bindingpartner and detectable entity that comprise the conjugate, but caninclude albumins, caseins, gelatin, polymeric stabilizers such aspolyvinylpyrrolidone or polyvinyl alcohol, or sugars like sucrose andtrehalose. In some embodiments, the conjugates are lyophilized or driedand are solubilized upon contact with the sample during operation of thedetection system.

In another embodiment of the invention, the one or more channels presenton the surface further contain a positive or negative control entity.The control entity can operate as a positive control for the detectionsystem. For instance, a detectable signal from the control entity canindicate that the liquid sample applied to the detection system hasreached the capture ligand(s) of the system (e.g. the fluidics of theradial flow paths are functioning properly). This function of thecontrol entity helps to eliminate false negatives due to improper flowof the sample through the detection system. In some embodiments, thecontrol entity is an immobilized control binding partner. In oneembodiment, the control binding partner is capable of specificallybinding to the unreacted conjugate (e.g. the binding partner ordetectable entity). By way of example, the control binding partner canbe an antibody that specifically binds to the binding partner ordetectable entity. Preferably, the detection signal is the same for boththe conjugate and control.

In another embodiment, the control binding partner is capable ofspecifically binding to an artificial component (e.g. control conjugate)that has been added to the sample. The artificially added controlconjugate can be supplied to the sample prior to sample application tothe detection system or it may be dried onto a porous material locatedin the sample port of the system. For instance, biotin coupled to adetectable entity (e.g. biotin conjugate) can be supplied to the sample.In this case, the control binding partner can be streptavidin, whichwould bind the artificially added biotin conjugate. In preferredembodiments, the detectable entity coupled to the artificially addedsubstance is the same as the detectable entity in the conjugate.

In one embodiment, the positive or negative control entity is positionedin each channel downstream of each capture ligand. See, e.g., FIG. 1. Insuch an embodiment, the control entity indicates proper fluid flow ineach radial flow path or channel. In another embodiment, the positive ornegative control entity is positioned at the end of a single flow pathdownstream of all capture ligands. See, e.g., FIG. 2. In thisembodiment, the control entity indicates whether the sample wastransported along the full-length of the flow path to the end of thedetection system. Control entities can be immobilized to surface,channel, or porous materials with methods similar to those describedherein for immobilizing capture ligands.

In some embodiments, the surface further contains at least one absorbingentity located downstream of the capture ligand. The absorbing entitycan be positioned at the end of a single flow path downstream of one ormore capture ligands FIG. 2). In another embodiment, two or moreabsorbing entities are positioned on the surface so as to separate twoor more flow paths (e.g., FIG. 1). For instance, a first absorbingentity can be positioned at the end of a first radial flow pathdownstream of a first capture ligand and a second absorbing entity canbe positioned at the end of a second radial flow path downstream of asecond capture ligand such that the first and second radial flow pathsdo not intermix. The one or more absorbing entities can supplement thefunction of the centrifugal force in moving fluids through the one ormore radial flow paths of the detection system. The absorbing entitiesalso function to remove excess fluid from other components of the systemand can help pull away unreacted (e.g. uncaptured) conjugate, thuspreventing an undesirable background noise at the capture ligands. Theabsorbing entity can be constructed from cellulose materials or the likeor can be a hygroscopic powder, polymer, or material. In a preferredembodiment, the absorbing entity is a cross linked polyacrylamidecopolymer that absorbs water (e.g., Waterlock®).

The detection system of the invention is preferably adapted for use witha centrifugal force. In one embodiment, the centrifugal force isoperably connected to the sample port so that when in operation itcauses a sample deposited in the sample port to move through the one ormore channels present on the surface and be in fluid contact with theone or more capture ligands. As used herein “operably connected” meansthat the centrifugal force is applied to the sample port such that thesample port and surface rotate about a fixed axis (e.g. vertical orhorizontal axis). The centrifugal force causes a fluid sample depositedin the sample port to flow radially outward along channels to theperiphery of the surface. The centrifugal force-driven movement allowsthe sample to interact with reagents and capture ligands deposited atvarious points along the flow paths. In embodiments in which eachchannel comprises a first and second flow path (i.e. dual flow paths),the applied centrifugal force causes the fluid sample to flow into bothflow paths. In some embodiments, a higher centrifugal force is requiredto move fluid through the second flow path of each channel as comparedto the centrifugal force required to move fluid through the first flowpath of each channel. In one embodiment, two different centrifugalforces are applied to the detection system at different times. Forinstance, a lower centrifugal force (i.e. low speed spin) is applied tothe system to move liquid sample from the sample port into the firstflow path of each channel to the capture ligands located on theperipheral edge of the surface. A second, higher centrifugal force (i.e.high speed spin) is subsequently applied to the system to move thesample through the second flow path of each channel to contact thecapture ligands located on the peripheral edge of the surface. Thus, thedelivery of reagents to the capture ligands of the detection system canbe delayed by employing a second flow path in each channel.

The applied centrifugal force can be from about 500 g to about 2,000 g,from about 800 g to about 1,600 g, or from about 1,000 g to about 1,200g. The detection system can be used with conventional centrifuges aslong as the appropriate centrifugal forces can be applied. In someembodiments, the detection system is adapted for use with the Piccolo®xpress and VetScan® analyzers available from Abaxis, (Union City,Calif.).

One embodiment of the analyte detection system of the invention isillustrated in FIG. 1. In this embodiment, the detection systemcomprises a surface, such as a rotor base or disc, containing aplurality of channels that are in fluid communication with a sampleport. Each channel contains a conjugate comprised of a binding partnerconjugated to a detectable entity that can specifically bind to a targetanalyte present in the sample. At the peripheral edge of each channel isa capture ligand capable of binding the analyte-conjugate complex. Theperipheral edge can optionally contain a control entity (control line)that indicates sufficient fluid flow through the system. The sample portmay optionally contain a blood separator material when the detectionsystem is used to analyze whole blood samples.

A liquid sample containing one or more analytes is deposited. In thesample port. A centrifugal force is applied to the surface and sampleport such that the surface and sample port spin about a fixed axis. Thefluid sample in the sample port flows radially outward through thechannels to the periphery of the surface (blue arrows). The sampleflowing through each radial flow path will pass through the conjugatewhere it will solubilize the dried conjugate. If the target analyte ispresent in the sample, the conjugate will bind to the target analyteforming a complex and continue to flow downstream to the capture ligand.The immobilized capture ligand present at the peripheral edge of thesurface will bind to and capture the target analyte-conjugate complex.Excess fluid containing unreacted conjugate (e.g. not bound to thetarget analyte) will pass through the region containing the captureligand and into the region containing the control entity (if present),which will capture some of the unreacted conjugate. The remaining samplewill be absorbed by one or more absorbing entities positioned downstreamof each capture ligand. The conjugates and capture ligands in eachradial flow path (channel) can bind to a different analyte in thesample, thereby allowing simultaneous detection of multiple analytes. Adetectable signal measured from each captured analyte-conjugate complexat the peripheral edge of each channel indicates that the particularanalyte is present in the sample.

FIG. 2 illustrates another embodiment of the analyte detection system ofthe invention. In this embodiment, the detection system comprises asurface (e.g. rotor base or disc) containing a single channel fluidcommunication with a sample port. The sample port contains multipleconjugates, each of which are comprised of a binding partner conjugatedto a detectable entity. The conjugates are capable of specificallybinding to different target analytes present in the sample. Theperipheral edge of the surface contains multiple capture ligands capableof binding particular analyte-conjugate complexes. A control entity(control line), which indicates sufficient fluid flow through thesystem, may optionally be positioned at the end of the peripheral flowpath upstream of the absorbing entity. Preferably, a water imperviousmaterial (block) is positioned between the end of the peripheral flowpath and the point where the channel delivers fluid to the peripheraledge of the surface. The block or dam can be constructed from plastic,silica plate, metal plate, or a laminated or coated material or otherwater impervious material. When the detection system is used foranalyzing whole blood samples, the sample port can optionally contain ablood separator material, which allows plasma from a blood sample topass into the system while retaining cellular material in the sampleport.

In this format, a sample containing one or more target analytes isdeposited in the sample port, which solubilizes and interacts with themultiple conjugates. If the particular analytes to which each of theconjugates bind is present in the sample, analyte-conjugate complexesare formed. A centrifugal force is applied to the sample port andsurface such that the surface and sample port spin about a fixed axis.The sample fluid containing the analyte-conjugate complexes flowsradially through the channel to the periphery of the surface (bluearrows). Capture ligands immobilized on the peripheral edge of thesurface will sequentially capture each of the analyte-conjugatecomplexes, Excess fluid sample is absorbed by an absorbing entitypositioned at the end of the peripheral flow path (e.g., downstream ofall capture ligands). A detectable signal measured from each capturedanalyte-conjugate complex at each of the peripheral sets of captureligand indicates that the particular analytes are present in the sample.

The present invention also includes kits comprising the analytedetection systems of the invention as disclosed herein. In oneembodiment, the kit comprises an analyte detection system andinstructions for using the system to detect one or more analytes in atest sample, wherein the detection system is adapted for use with acentrifugal force and comprises a sample port and a surface, saidsurface containing at least one channel, said channel comprising animmobilized capture ligand capable of specifically binding to an analytein a sample, and wherein the sample port is in fluid communication withsaid at least one channel. In certain embodiments, the surface is in arotor or disc format capable of use with conventional centrifuges. Thekit can further include means for collecting biological samples orextraction buffers for obtaining samples from solid materials, such assoil, food, and biological tissues.

The present invention also encompasses a method for detecting an analytein a sample. In one embodiment, the method comprises adding the sampleto the sample port of a detection system disclosed herein, applying acentrifugal force to the system, and detecting the binding of theanalyte to the capture ligand. In some embodiments, the detection stepcomprises measuring an optical signal from a captured analyte-conjugatecomplex. The invention provides a method for detecting multiple analytespresent in a single sample simultaneously. The methods can providequalitative and or quantitative analysis of the detected analytes.

A sample can be any type of liquid sample, including biological samplesor extracts prepared from environmental or food samples. In a preferredembodiment, the sample is a biological sample. Biological samplesinclude, but are not limited to, whole blood, plasma, serum, urine,pleural effusion, sweat, bile, cerebrospinal fluid, fecal material,vaginal fluids, sperm, ocular lens fluid, mucous, synovial fluid,peritoneal fluid, amniotic fluid, biopsy tissues, saliva, and cellularlysates. The biological sample can be obtained from a human subject oranimal subject suspected of having a disease condition, such as cancer,infectious diseases (e.g., viral, bacterial, parasitic or fungalinfections), cardiovascular disease, autoimmune etc. The biologicalsample can also be obtained from a healthy subject (e.g. human oranimal) undergoing a routine medical check-up.

Any type of target analyte can be detected using the methods and systemsof the present invention. An “analyte” refers to any substance capableof being bound by a capture ligand or binding partner of the conjugatesdisclosed herein. An analyte encompasses derivatives or metabolites ofthe compound of interest. In some embodiments, the analytes areassociated with infectious diseases in both humans and animals. In otherembodiments, the analytes are markers of a particular physiological orpathological condition, A target analyte can be a protein, peptide,nucleic acid, hapten, or chemical.

In certain embodiments, the analyte is a pathogenic antigen or antibodyto a pathogenic antigen. For instance, the pathogenic antigen can be aviral antigen (e.g., feline leukemia virus, canine parvovirus, foot andmouth virus, influenza virus, hepatitis a, b, c virus, HIV virus, humanpapilloma virus, epstem barr virus, rabies virus, etc.), a bacterialantigen (e.g., Ehrlichia, Borellia, Anthrax, Salmonella, Bacillus,etc.), a fungal antigen, or parasitic antigen (e.g., canine heartworm,Giardia lamblia, plasmodium falciparum, african trypanosomiasis,trypanosoma brucei, etc.). In other embodiments, the analyte is adisease-related antigen or antibody to a disease-related antigen.Disease-related antigens include, but are not limited to, cancer-relatedantigens (e.g., PSA, AFP, CA125, CA15-3, CA19-9, CEA, NY-ESO-1. MUC1,GM3, GD2, ERBB2, etc.), cardiovascular disease-related antigens (e.g.,troponin, CRP, CKMB, fatty acid binding protein, etc.), or autoimmunedisease-related antigens (e.g., auto-antibodies). In certainembodiments, the analyte is a inflammatory antigen (e.g., C-reactiveprotein, MRP14, MRP8, 25F9, etc.). In other embodiments, the analyte isa pregnancy-related antigen (e.g., a fetal antigen).

Detection of binding of the analyte to the capture ligand comprisesobserving or measuring the signal from the detectable entity of capturedanalyte-conjugate complexes. In some embodiments, the signal is aspectral shift (e.g. color change). In other embodiments, the signal isa fluorescent signal. The detection signal corresponding to the presenceof any captured analyte-conjugate complexes at the capture ligands canbe detected visually or by means of an instrument. In one embodiment,the signal is detected by measuring a change in absorbance of thesignal. Commercial instruments capable of detecting spectral shifts orchanges in fluorescence can be used to measure the detection signal fromcaptured analyte-conjugate complexes of the detection systems. Suchinstruments include “strip readers” and are known to those skilled inthe art. The means for detecting a signal from the detectable entity inthe captured analyte-conjugate complexes can be located in the sameinstrument that applies the centrifugal force. Quantitative analysis ofthe analytes can be achieved by measuring spectral shifts or absorbancechanges and comparing the shifts/changes to those obtained with knownconcentrations of analytes. For instance, in one embodiment,concentration of the target analyte in a sample can be calculated fromthe intensity of the optical signal (e.g. intensity of color change) atthe capture ligand. Detection of a signal from the control entity of theanalyte detection systems of the invention is indicative of proper fluidflow through the device. Detection of a signal from the regionscontaining capture ligands of the analyte detection systems of theinvention is indicative of the presence of target analyte in the sample(e.g. positive sample). Similarly, absence of a signal from the regionscontaining capture ligands of the analyte detection systems of theinvention is indicative of the absence of target analyte in the sample(e.g. negative sample).

It is understood that the disclosed invention is not limited to theparticular methodology, protocols and materials described as these canvary. It is also understood that the terminology used herein is for thepurposes of describing particular embodiments only and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. A system comprising a sample port and asurface, wherein the surface comprises a plurality of channels arrangedradially on the surface and in fluid communication with a continuouschannel arranged around the peripheral edge of the surface, each of saidplurality of channels containing an immobilized capture ligandpositioned at the peripheral edge of the surface and a conjugate locatedwithin each of the plurality of channels such that the conjugate islocated upstream of the immobilized capture ligand; wherein theconjugate comprises a binding partner conjugated to a detectable entity,wherein said conjugate is capable of binding to an analyte in a sampledeposited in the sample port to form a complex that is captured by thecapture ligand, wherein the sample port is in fluid communication withsaid plurality of channels, and wherein spinning the surface and thesample port about a fixed axis generates a centrifugal force that causesthe sample deposited in the sample port to flow through the plurality ofchannels and into the continuous channel, such that the sample contactsthe conjugate and the capture ligand.
 2. The system of claim 1, whereinthe detectable entity is a metallic nanoparticle or metallic nanoshell.3. The system of claim 2, wherein the detectable entity is selected fromthe group consisting of gold nanoparticles, silver nanoparticles, coppernanoparticles, platinum nanoparticles, cadmium nanoparticles, compositenanoparticles, gold hollow spheres, gold-coated silica nanoshells, andsilica-coated gold shells.
 4. The system of claim 1, wherein thedetectable entity is an enzyme.
 5. The system of claim 4, wherein theenzyme is selected from the group consisting of alkaline phosphatase,horseradish peroxidase, beta-galactosidase, beta-lactamase, luciferase,myeloperoxidase, and amylase.
 6. The system of claim 1, wherein thesurface further contains at least one absorbing entity locateddownstream from the capture ligand.
 7. The system of claim 1, whereinsaid conjugate in each channel specifically binds to a different analytein the sample.
 8. The system of claim 1, wherein the sample portcomprises a blood separator material that allows plasma from a bloodsample to pass into said plurality of channels while retaining cellularmaterial from the blood sample in the sample port.
 9. The system ofclaim 1, wherein at least one of said channels further contains apositive or negative control entity.
 10. The system of claim 1, whereinthe system comprises a first flow path comprising said plurality ofchannels, and the system further comprises a second flow path, whereinsaid second flow path separates from the first flow path at a firstpoint downstream from the sample port and rejoins the first flow path ata second point between said first point and a region comprising saidimmobilized capture ligand, and wherein said first flow path containssaid immobilized capture ligand and said conjugate.
 11. The system ofclaim 10, wherein said second flow path comprises a substrate region,said substrate region comprising a substrate entity capable ofinteracting with the detectable entity in the conjugate to produce adetectable signal.
 12. The system of claim 10, wherein said second flowpath comprises a substrate region, said substrate region comprising asubstrate entity capable of interacting with the detectable entity inthe conjugate to amplify the signal from the detectable entity.
 13. Thesystem of claim 12, wherein the detectable entity is a gold nanoparticleand the substrate entity is silver nitrate, silver acetate, or silvercitrate.
 14. The system of claim 12, wherein the detectable entity is ametallic nanoparticle and the substrate entity is the peroxidasereaction product of 3,3′,5,5′-Tetramethylbenzidine (TMB) or an alkalinephosphatase reaction product of BCIP.
 15. The system of claim 10,wherein the first flow path provides a faster flow through than thesecond flow path when said centrifugal force is applied to said at leastone channel.